Method for producing non-infectious recombinant picornavirus particles

ABSTRACT

The invention relates to a process for recombinantly preparing picornavirus particles, in particular hepatitis A virus particles, their precursors and particles which are derived therefrom, with a structural protein precursor molecule (P1-2A or P1) and the corresponding P3 region (3ABCD) being coexpressed in cis or in trans.

This application claims benefit of PCT/DE98/00879, filed Mar. 26, 1998,now abandoned, which claims benefit of DE 197 12 899.8, filed Mar. 27,1997, which issued as German Patent No. 197 12 899 on Dec. 10, 1998.

The invention relates to a process for recombinantly preparingpicornavirus particles, in particular hepatitis A virus particles, theirprecursors and particles which are derived therefrom.

It is known from the state of the art to prepare picornavirus strains byculturing in eukaryotic cells. Such culturing is particularlyinefficient in the case of particular members of the Picornaviridaefamily, for example the hepatitis A virus. A further disadvantage isthat the picornaviruses which are cultured in the eukaryotic cells areinfectious. They have to be chemically inactivated before being used,for example as a dead vaccine. Residues of the substance used for theinactivation, for example formaldehyde, may elicit allergic reactionsand alter the antigenicity of the virus particle.

Stapelton, J. T. et al. 1990 have described (in Hollinger, F. B. et al.:Viral hepatitis and liver disease, 50-54) a more efficient method whichcan be used, in particular, for preparing empty, recombinant hepatitis Avirus particles. The particle yields which are achieved by this methodare likewise not adequate for commercial applications.

It is furthermore known from the state of the art that polyproteinprocessing is mediated by 3CD, 3C and 2A in the case of the enterovirusand rhinovirus genera, by L-proteinase and 3C in the case of thecardiovirus and apthovirus genera, and by 3C in the case of thehepatovirus genus. It is not known from the state of the art whetherother nonstructural proteins are involved in the formation of emptyvirus particles.

The object of the present invention is to specify a process whicheliminates the disadvantages of the state of the art. The aim is, inparticular, to make available a more efficient process.

According to the invention, a structural protein precursor molecule andthe corresponding P3 region (3ABCD) are coexpressed, in cis or in trans,for the purpose of recombinantly preparing picornavirus particles, inparticular hepatitis A virus particles, their precursors and particleswhich are derived therefrom. The part proteins 2B and 2C areadvantageously not expressed in this connection. One of the advantagesof the novel process is that the inhibitory effects associated withexpressing the part proteins 2B and 2C are eliminated and use is onlymade of the minimum sequences which are required for efficientlysynthesizing empty picornavirus particles. This improves the efficiencyof the process, on the one hand, and excludes the possibility ofpicornaviral replication, on the other hand. Using the complete P3region markedly increases the efficiency of the synthesis of emptypicornavirus particles, or their precursor molecules, as compared withonly using the 3C and/or 3CD proteinases.

Advantageously, the structural protein precursor molecule possessessequences encoding P1 or P1-2A. The P1 structural protein precursormolecule is in particular used in the case of the enterovirus andrhinovirus genera. In the case of the cardiovirus, aphthovirus andhepatovirus genera, use is preferably made of the structural proteinprecursor molecule which possesses the sequence encoding P1-2A.

A particularly efficient preparation of empty hepatitis A virusparticles can be achieved when the VP1-2A cleavage site at amino acidposition 273/274 is occupied by a glutamate/serine pair or aglutamine/serine pair, with the indication of the amino acid positionrelating to the sequence of the attenuated strain HM175 (in accordancewith Cohen, J. I. et al. 1987, Journal of Virology 61:3035-3039).

When the structural protein precursor molecule and the P3 proteins areexpressed in cis, there is equimolar expression of the two genomeregions. This enables empty hepatitis A virus particles to be preparedefficiently. An even more efficient preparation of hepatitis A virusparticles can be achieved by optimizing the molar ratio of thestructural protein precursor molecule and the P3 proteins by means ofexpressing in trans. The coexpression in trans can be carried out bycoexpressing the structural protein precursor molecule and the P3 regionwhen they are encoded on separate vectors. This coexpression can also becarried out using a bicistronic expression vector or a bicistronic mRNA.In this context, at least two promoter sequences or translationinitiation sequences which are of differing efficiencies areadvantageously located in front of the P3 cistron and the structuralprotein cistron. This makes it possible to adjust the molar ratio of thestructural protein precursor molecule and the corresponding P3 regionoptimally for achieving efficient synthesis of empty picornavirusparticles.

It has proved to be advantageous for the expression of the proteins ofthe P3 region to be controlled negatively in cis by means of cloningsequence elements of the 5′ non-translated region (=5′ NTR) in front ofthe sequences encoding the P3 region. The negative control of theexpression of the proteins of the P3 region contributes, when thisregion is being coexpressed together with the structural proteinprecursor molecule, to the two components being expressed in an optimalratio.

The negatively controlling sequence elements employed can expediently bethe sequences which are located in front of the internal ribosomebinding site of the 5′ NTR. Particularly in the case of the hepatitis Avirus, it has proved to be advantageous for the sequence elements of the5′ NTR to be the 24 or 45 5′ terminal nucleotides of the 5′ NTR of thehepatitis A virus genome, or to be derived therefrom.

According to another organizational feature of the process, thecoexpression is carried out in eukaryotic or prokaryotic cells or in thecorresponding cell extracts, with the sequences which are to becoexpressed being cloned 3′ of a eukaryotic or of a prokaryoticpromoter. The Semliki forest virus expression system (Liljeström P.,Garoff H., Bio/Technology 9, 1356-1361) or the baculovirus expressionsystem (Bishop D H L, 1990, 62-67, Current Opinion in Biotechnology)can, for example, be used in this context.

The invention furthermore encompasses a kit or a combination of meansfor carrying out the above-described process. Finally, the picornavirusparticles according to the invention can be used for preparing amedicament or vaccine or for preparing galenics or diagnostic agents.

The invention is explained in more detail below in the drawing and withthe aid of implementation examples.

FIG. 1 shows the genomic organization of the hepatitis A virus,

FIG. 2 shows the Semliki forest virus expression constructspSFV3-HM/HM-P1-2A(E/S) and pSFV1-HM-P3,

FIG. 3 shows the expression construct pGEM2-HAV-Δ2BC, and

FIGS. 4a and b show the detection of empty, recombinantly producedhepatitis A virus particles.

FIG. 1 depicts the genomic organization of the hepatitis A virus. Theinternal ribosome binding site (IRES) which is located in the 5′non-translated region (5′ NTR) mediates the initiation of polyproteinsynthesis. The polyprotein encodes the structural proteins (VP4-VP1) andthe nonstructural proteins (2A-3D). The former form the virus coat; thelatter assume various functions in the lifecycle of the virus.

FIG. 2 shows pSFV3-HM/HM-P1-2A(E/S), which encodes the P1-2A structuralprotein precursor molecule having a Glu/Ser-VP1-2A cleavage site, andpSFV1-HM-P3, which encodes the complete P3 region of the attenuated HAVstrain HM175. After transfecting in-vitro transcribed and capped RNAinto suitable host cells, the P3 proteins mediate their own proteolyticprocessing and the proteolytic processing of the P1-2A structuralprotein precursor molecule. In addition to this, they mediate theefficient formation of empty hepatitis A virus particles.

The expression construct possessing a T7 promoter, which is depicted inFIG. 3, encodes an HAV polyprotein from which the 2BC region has beenalmost completely deleted. The reading frame of the part polyprotein hasnot been destroyed. After transfecting into suitable cells and makingavailable T7 RNA polymerase, the T7 promoter mediates the transcriptionof the partially deleted viral genome. The remains of the 2BC regionmake possible the 3C-mediated polyprotein cleavage between 2A and Δ2Band also between Δ2C and 3A and consequently the preparation ofauthentic P1-2A and P3. The P3 proteins mediate the subsequentproteolytical cleavages of the recombinant proteins and enable emptyhepatitis A virus particles to be formed efficiently.

FIGS. 4a and 4 b depict the profiles of the sucrose gradients of theconstructs shown in FIG. 2 and FIG. 3 after ultracentrifugation. Theviral antigen was detected in an ELISA using the neutralizing monoclonalantibody K2-4F2. The major portion of the K2-4F2-reactive viral antigenmigrated into the 20% (w/w) sucrose fraction. This gives a sedimentationconstant of about 90S. Consequently, the recombinantly producedhepatitis A virus antigen corresponds structurally and immunologicallyto empty hepatitis A virus particles.

The following parameters were used in the ultracentrifugation: runningtime=75 minutes; centrifuge speed=48,000 rpm; temperature=5° C.; rotorSW65 (Beckmann); gradient composition: 5-30%(w/w) sucrose in 10 mM tris,pH 7.3. The gradient was calibrated using an HAV-infected cell extract.

EXAMPLES

1. The Production of Empty Hepatitis A Virus Particles by CoexpressingP1-2A and P3 in Trans Using the Semliki Forest Virus Expression System(Liljeström, P., Garoff, H., Bio/Technology 9, 1356-1361).

The 2.7 kb MscI/XhoI fragment from pEXT7-HM/HM-P1-2A(E/S) and the vectorpSFV3 (Gibco-BRL, Bethesda, USA), which was linearized with BamHI anddephosphorylated, were used for cloning pSFV3-HM/HM-P1-2A(E/S) asdepicted in FIG. 2. The single-stranded protruding ends were removed,prior to the ligation, by means of a Klenow filling-in reaction. Theplasmid pSFV3-HM/HM-P1-2A(E/S) encodes the P1-2A of the (attenuated) HAVstrain HM175, together with 5 N-terminal foreign amino acids, with theVP1-2A cleavage site at the VP1 amino acid position 273/274 being aglutamate/serine pair (E/S). Translation initiation takes place, in acap-dependent manner, by way of a start codon which is located in aKozak consensus sequence (Kozak, M. 1989. J. Cell. Biol. 108:229-225).

For cloning pSFV1-HM-P3 (see FIG. 2), pSFV1 (Gibco-BRL, Bethesda, USA)was first of all linearized with BamHI and the single-strandedprotruding ends were filled in by means of a Klenow polymerase reaction.The P3 genomic region (bases 4996-7409) was amplified by PCR using the“P3 sense” (5′- ATG GTC CGG AGC ACC ATG GGA ATT TCA GAT GAT GAT AAT GATAG-3′SEQ ID NO. 3.) and “P3 antisense” (5′-T TTA GCT AGC TCA TGA AAG GTCACA AAT GAA ACA CTG GTC A-3′) primers and pT7-HAV1 as the template. ThePCR fragment was then cut with Kpn21/NHeI, after which the protrudingsingle-stranded regions were filled in using Klenow polymerase.pSFV1-HM-P3 was cloned by inserting this fragment into thedephosphorylated pSFV1 vector fragment. pSFV1-HM-P3 encodes the entireP3 region of the attenuated HAV strain HM175, with a methionine residuebeing encoded as an additional N-terminal amino acid. Translationinitiation is mediated by a Kozak translation initiation sequence.

The SpeI-linearized plasmids pSFV3-HM/HM-P1-2A(E/S) and pSFV1-HM-P3 areused as the templates for the in-vitro transcription of capped RNA(Current Protocols in Molecular Biology, Supplement 25, John Wiley &Sons, Inc.). Equal quantities of the unpurified SFV3-HM/HM-P1-2A(E/S)and SFV1-HM-P3 in-vitro transcription mixtures are mixed together and ineach case transfected by electroporation into 2×10⁶ COS7 cells (fortransfection parameters, see Wilk, T. et al. 1992, Virus Genes6:229-246). The cells are then sown on 30 cm² cell culture dishes andincubated in a cell culture incubator for 48 hours. The recombinantlyproduced empty virus particles are detected, for example, byultracentrifuging in a continuous 5-30%(w/w) sucrose gradiant. For this,the fractions of the sucrose gradient are analyzed by an “enzyme-linkedimmunosorbent assay” (ELISA) which is based on the neutralizingmonoclonal antibody K2-4F2 (MacGregor, A. W. et al. 1983, J. Clin.Microbiology 18: 1237-1243). The recombinantly produced antigenpossesses a sedimentation constant of about 90S and, as is evident fromFIG. 4a, corresponds structurally and immunologically to empty hepatitisA virus particles.

2. Production of Empty Hepatitis A Virus Particles by Coexpressing P1-2Aand P3 in cis in the vaccinia T7 Expression System (Fuerst, T. R. et al.1986, Proc. Natl. Acad. Sci. USA. 83:8122-8126).

The sequence region of the plasmid p5′P2P3-3′.18f (Zhang, H. et al.1995, virology 212:686-697) which encodes the HAV genome of anattenuated HAV strain is first of all placed under the control of a T7promoter. To do this, the 7.5 kb HindIII/SphI fragment is inserted intopGEM2 which has been cut in a compatible manner.

After that, the 2BC genome region is deleted from the resulting cDNAconstruct pGEM2-HAV. For this, the VP1-Δ2B-encoding region is amplifiedby PCR using the “VP1 sense” (5′-GGG GTA CCC ACA ACC ATG GTT GGA GAT GATTCT GGA GGT-3′SEQ ID NO. 3) and “Δ2B antisense” (5′-CC GGT ACC CTC AGTATA AAA GAA AGA AAT TTA GGC-3′SEQ ID NO. 2) primers and pGEM2-HAV as thetemplate. The resulting DNA fragment is cut with KpnI and thesingle-stranded protruding end is digested off by means of a mung beannuclease reaction; the fragment is then cut with SacI. This fragment isligated into pGEM2-HAV which has been cut with EcoRI/SacI SacI and whoseEcoRI cleavage site has been filled in using Klenow polymerase. Theresulting construct, pGEM2-HAV-Δ2BC (FIG. 3), is transfected into COS7cells by means of lipofection. Expression is initiated by infecting withvaccinia T1 virus. 24 hours after the infection, the cell extract isanalyzed in an analogous manner to that described in Example 1. Therecombinantly produced antigen has a sedimentation constant of 90S and,as is evident from FIG. 4b, corresponds structurally and immunologicallyto empty hepatitis A virus particles.

4 1 44 DNA Artificial Sequence primer 1 atggtccgga gcaccatggg aatttcagatgatgataatg atag 44 2 40 DNA Artificial Sequence primer 2 tttagctagctcatgaaagg tcacaaatga aacactggtc 40 3 39 DNA Artificial Sequence primer3 ggggtaccca caaccatggt tggagatgat tctggaggt 39 4 35 DNA ArtificialSequence primer 4 ccggtaccct cagtataaaa gaaagaaatt taggc 35

What is claimed is:
 1. A method for recombinantly producingnon-infectious Hepatitis A virus particles comprising expressing anucleic acid encoding a Hepatitis A virus structural protein precursormolecule and a nucleic acid encoding a Hepatitis A virus P3 region in acell, wherein said cell does not express Hepatitis A virus proteins 2Band 2C.
 2. The method of claim 1, wherein said nucleic acids areexpressed in cis orientation.
 3. The method of claim 1, wherein saidnucleic acids are expressed in trans orientation.
 4. The method of claim3, wherein said nucleic acids are expressed from a bicistronicexpression vector.
 5. The method of claim 1, wherein said cell iseukaryotic.
 6. The method of claim 1, wherein said structural proteinprecursor molecule is P1.
 7. The method of claim 1, wherein saidstructural protein precursor molecule is P1-2A.
 8. The method of claim1, wherein positions 273 and 274 of P1-2A are glutamine and serine,respectively.
 9. The method of claim 1, wherein positions 273 and 274 ofP1-2A are glutamate and serine, respectively.
 10. The method of claim 1,wherein said nucleic acid encoding said P3 region comprises negativecontrol elements.
 11. The method of claim 10, wherein said negativecontrol elements comprise 5′ terminal nucleotides of the 5′non-translated region of said P3 region.
 12. The method of claim 1,wherein said nucleic acids comprise a promoter.
 13. The method of claim12, wherein said promoter is a CMV promoter.
 14. The method of claim 12,wherein said promoter is a T7 promoter.
 15. The method of claim 1,wherein said P3 region encodes 3A, 3B, 3C, and 3D.
 16. A kit forproducing recombinant non-infectious Hepatitis A virus particles, saidkit comprising: a) a nucleic acid encoding a Hepatitis A virusstructural protein precursor molecule; and b) a nucleic acid encoding aHepatitis A virus P3 region.